The overall goal of this proposal is to investigate the mechanism by which Insulin Receptor Substrate-2 (IRS- 2) regulates tumor cell metabolism to promote breast cancer progression and metastasis. The IRS proteins are adaptor molecules that are recruited to activated cell surface receptors where they organize signaling complexes to initiate downstream signaling cascades. Many of the receptors that signal through the IRS proteins have been implicated in breast cancer, which highlights the importance of understanding how these cytoplasmic molecules impact tumor cell function. In humans, both IRS-1 and IRS-2 are expressed in normal and benign breast tissue, DCIS and invasive breast cancer. However, these adaptor proteins play divergent roles in mammary tumor progression. Specifically, metastasis is significantly impaired in the absence of IRS-2, but it is enhanced in tumor cells that lack IRS-1, but have increased IRS-2 expression and activation. In vitro, signaling through IRS-2 promotes tumor cell migration, invasion and survival. Although functional differences between IRS-1 and IRS-2 have been identified, the mechanism(s) by which these highly homologous proteins regulate distinct cellular outcomes in response to common stimuli remains unknown. To predict the consequences of the relative expression of IRS-1 and IRS-2 for breast cancer patients and to determine how their expression and function could be manipulated to interfere with breast cancer progression, additional mechanistic studies of these adaptor proteins are needed. Recent studies in the applicant's lab reveal that IRS-2, but not IRS-1, plays an essential role in regulating glycolysis to promote tumor cell viability and invasion. Furthermore, IRS-2 expression is increased by hypoxia, which impinges upon the metabolic status of tumors to promote aggressive behavior. The overall hypothesis to be examined in this proposal is that IRS-2 is an essential hypoxia-responsive gene that promotes breast cancer progression and metastasis through the regulation of tumor cell metabolism. To investigate this hypothesis, the applicant proposes to: 1) Determine the mechanism by which IRS-2 regulates metabolism in breast carcinoma cells. The hypothesis that IRS-2 enhances glucose uptake and promotes aerobic glycolysis through the targeted activation of PI3K/mTOR signaling to regulate the surface expression of GLUT1 will be examined; and 2) Investigate the mechanism by which hypoxia enhances IRS-2 expression and define the functional significance of this regulation for breast cancer metabolism and progression. The hypothesis that IRS-2 expression is increased in hypoxic environments at the level of gene transcription to sustain breast carcinoma cell metabolism and to promote tumor progression and metastasis will be examined. PUBLIC HEALTH RELEVANCE: Metastasis is the leading cause of death for cancer patients and novel approaches to impede or treat metastatic tumors are needed. Tumor cells that develop metabolic self-sufficiency can survive in stressful environments that lack oxygen and other essential nutrients for energy production, and this creates a selective pressure to progress to invasive and metastatic disease. In this proposal, one mechanism by which tumor cells regulate their metabolism to promote metastasis will be examined with the long term goal of developing new therapies to benefit cancer patients with metastatic tumors.